Epithelial-mesenchymal transition (EMT) has been shown to play an important role in the fibroses of multiple organs and tissues, including the ocular lens, where it contributes to both anterior subcapsular cataracts (ASC) and posterior capsular opacification (PCO), also known as secondary cataract. Increased proliferation of lens epithelial cells (LECs), and EMT of LECs into myofibroblasts, involving a loss of the cell-cell adhesion molecule E-cadherin and an induction in ?-smooth muscle actin (?SMA) expression are early events in both ASC and PCO. Transforming growth factor beta (TGF?) is a pleotropic morphogen that has been shown to induce the EMT of LECs and subsequent formation of ASC, as well as, PCO. Using a previously developed rat lens culture model in which exogenous TGF? induces ASC we have shown that treatment with inhibitors to the matrix metalloproteinases (MMP), specifically MMP-2 and MMP-9, suppresses TGF? -induced cataractous changes, including EMT. Studies from the previous grant period further show that these two MMPs likely work cooperatively and/or redundantly in the development of these cataracts. For example, using a model of ASC involving the delivery of Ad TGF? to the eye we have shown that MMP-9 KO mice develop cataracts, albeit they are delayed compared to wild-type mice. Thus, inhibiting both MMPs may be required to prevent EMT and subsequent cataractogenesis. The potential mechanism by which these MMPs mediate EMT and cataract formation was identified during the previous funding period and involves disruption of E-cadherin. Preliminary data suggests that disruption and shedding of E-cadherin results in downstream signaling events linked to EMT including nuclear translocation of ?-catenin and the myocardin-related transcription factor (MRTF-A). However, the requirement for these signaling intermediates in ASC and PCO and how MMPs are involved is not known. In the current application we investigate these TGF? -mediated signaling pathways using multiple ex vivo and in vivo models of ASC and PCO. In addition, we outline experiments that will directly determine the unique and/or cooperative roles of MMP-2 and MMP-9 in ASC formation. Ultimately, our goal is to define the TGF? - mediated pathways controlling EMT and fibrosis in ASC and PCO in order to design therapeutics for mitigating these diseases. PUBLIC HEALTH RELEVANCE: Loss of transparency of the lens, or cataract, is the leading cause of blindness worldwide despite the availability of effective surgery in developed countries. Extracapsular cataract extraction is the most frequently performed surgical procedure in North America, costing over 3.5 billion dollars each year, and can frequently lead to complications such as the development of secondary cataract (posterior capsular opacification (PCO). Thus, an understanding of the cellular and molecular mechanisms regulating the normal and pathological differentiation of the lens is necessary in order to develop therapeutic strategies for the treatment and/or prevention of cataracts. In the proposed research we will investigate the cell signaling mechanisms responsible for two fibrotic cataracts, anterior subcapsular cataracts (ASC) and PCO and further determine how these mechanisms can be inhibited. Specifically we will focus on those pathways mediated by transforming growth factor beta (TGF?) and the matrix metalloproteinases (MMPs) since both of have been implicated in these cataracts. Ultimately, it is hoped that the data obtained from the proposed studies will lead to therapeutic strategies for mitigating cataract formation.